The major function of the link proteins (LP) of proteoglycan (PG) aggregates appears to be to stabilize the aggregation of proteoglycan monomers with hyaluronic acid (HA). Such aggregates form ordered structures with collagen and together these form the majority of the macromolecular species in cartilege. We propose to complete the determination of the primary structure of cartilage link protein (LP) and outline the locations of some portions of the sequence with respect to exposed functional groups of the molecule. To achieve this, link protein from both chick sternal cartilage and rat chondrosarcoma will be digested utilizing specific proteases (Staphylococcal V8 protease, trypsin, chymotrypsin and clostripain). Peptides which contain disulfide bonds will be prepared and digested to obtain peptides with single disulfide bonds. These will be separated by high performance liquid chromatography (RP-HPLC) and analyzed for their amino acid content and NH2-terminal sequence. The positions of disulfide bonds will be located in the known amino-acid sequence of LP. Peptides generated during the course of amino-acid sequence determination will be investigated for the possession of functional and/or exposed groups. This will be achieved by assaying fragments for their ability to bind either proteoglycan, hyaluronic acid or both macromolecules together using a combination of solid phase procedures and gel filtration. Both existing and new monoclonal antibodies will similarly be used to delineate exposed regions of the link protein which can be correlated with the effect of the antibodies on the aggregation of PG-LP-HA ternary complex to obtain a description of the external and/or functional portions of LP. Limited proteolysis or differential labelling of LP binary and ternary complexes will similarly outline regions of LP associated with the formation of macromolecular complexes. By this means we hope to increase the body of data pertaining to the function of structural proteins of cartilage and their mode of degradation in both normal and degenerative joint disease.